Characterization of Humanized Anti-TAC, an Antibody Directed Against the Interleukin 2 Receptor, Using Electrospray Ionization Mass Spectrometry by Direct Infusion, LC/MS, and MS/MS

Abstract

Characterization of a humanized monoclonal antibody (Hu-anti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillary reversed-phase liquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis to confirm the sequence and to determine the sources of heterogeneity in Hu-anti-TAC. The MS analysis was performed on disulfide-reduced and trypsin-digested samples of the antibody. Two forms of diantennary carbohydrate structures were identified and found to be consistent with those reported for the human IgG1 framework. The analysis demonstrated that the N-terminus was modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-terminal lysine residue and was confirmed by mass calculations of tryptic peptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of microgram samples of high molecular weight proteins such as antibodies.

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

“…Although antibodies have structural similarities, their post-translational modifications on heavy and light chains require constant monitoring from cell culture to final formulated products. Thus, it is obvious that the analysis of intact therapeutic monoclonal antibodies by reversedphase (RP) high-performance liquid chromatography (HPLC) online with mass spectrometry (LC/MS) would dramatically reduce sample preparation and data interpretation and also minimizes putative modifications that may occur during sample preparation, such as during peptide mapping experiments after enzymatic digestion [2].…”

“…However, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) [7,8], ESI orthogonal-TOF [9,10] and ESI quadrupole [2,[11][12][13] mass spectrometers have been the instruments of choice for MAbs and macromolecules, largely due to the large m/z range of the TOF and high ion transmission efficiency of the quadrupole mass analyzers. Unfortunately, on-line RP-HPLC has not accompanied these previous studies of intact monoclonal antibodies.…”

Mass Spectrometric Characterization of the β-Subunit of Human Chorionic Gonadotropin

“…HPLC/MS combined with di †erent enzymatic reactions has been used successfully for the characterization of a variety of natural and recombinant glycoproteins. 23,32 In this study, we report the use of mass spectrometry to characterize the b-subunit of hCG. This should provide a foundation for the development of a forensically robust method for hCG detection and for rapid characterization of candidate hCG protein standard preparations.…”

Determination of the origin of the N‐terminal pyro‐glutamate variation in monoclonal antibodies using model peptides

“…One common post-translational modification observed in monoclonal antibodies is a cyclized N-terminal glutamine (Beck et al, 2005;Gadgil et al, 2006;Lewis et al, 1994;Roberts et al, 1995;Werner et al, 2005) when either the heavy and/or light chain sequences begin with glutamine. This reaction illustrated in Figure 1 involves the cyclization of the N-terminal amine and the subsequent loss of NH 3 (À17 Da).…”